Preparation of One-Emission Nitrogen-Fluorine-Doped Carbon Quantum Dots and Their Applications in Environmental Water Samples and Living Cells for ClO− Detection and Imaging

Hypochlorite (ClO−) has received extensive attention owing to its significant roles in the immune defense and pathogenesis of numerous diseases. However, excessive or misplaced production of ClO− may pose certain diseases. Thus, to determine its biological functions in depth, ClO− should be tested in biosystems. In this study, a facile, one-pot synthesis of nitrogen-fluorine-doped carbon quantum dots (N, F-CDs) was developed using ammonium citrate tribasic, L-alanine, and ammonium fluoride as raw materials under hydrothermal conditions. The prepared N, F-CDs demonstrate not only strong blue fluorescence emission with a high fluorescence quantum yield (26.3%) but also a small particle size of approximately 2.9 nm, as well as excellent water solubility and biocompatibility. Meanwhile, the as-prepared N, F-CDs exhibit good performance in the highly selective and sensitive detection of ClO−. Thus, a wide concentration response range of 0–600 μM with a low limit of detection (0.75 μM) was favorably obtained for the N, F-CDs. Based on the excellent fluorescence stability, excellent water solubility, and low cell toxicity, the practicality and viability of the fluorescent composites were also successfully verified via detecting ClO− in water samples and living RAW 264.7 cells. The proposed probe is expected to provide a new approach for detecting ClO− in other organelles.


Introduction
Sodium hypochlorite at concentrations between 10 −5 and 10 −2 mol/L is widely used as a household bleach and disinfectant [1,2]. ClO − is an important type of reactive oxygen species that is widely used in the antimicrobial immunity of living systems [3,4]. It is extensively used in daily life as disinfectant and household cleaning agent for water treatment [5]. However, an excessive level of reactive oxygen species may cause certain pathological problems such as tissue aging, chronic infammatory diseases, and bladder cancer [4,6]. Terefore, an efective method detecting ClO − in environmental water samples and living cells should be established.
Currently, various approaches have been developed to detect and quantify hypochlorite, such as coulometric [7], chemiluminescence [8], potentiometric [9], and colorimetric methods [10]. Although these methods ofer unique advantages, they still possess several limitations, including special equipment, tedious operation, complicated sample preparation, and long time. Compared with other analytical methods, fuorescent probes ofer inherent benefts due to their high sensitivity [11], excellent specifcity [12], simple manipulation [13,14], low cost, short analytical times [15], and deep bioimaging capacity [16], which beneft application strategies for in vitro assays and in vivo imaging studies. For example, McCarroll [17] reported a pH-dependent probe to detect HOCl. Furthermore, certain HOCl probes can monitor HOCl in living cells. Duan et al. [18] proposed a hepatoma-specifc probe for examining HOCl, and Yuan et al. [19] developed two-photon fuorescent probes for HOCl imaging in mitochondria and lysosomes. Although the precursors play a vital role in improving the quantum yield and property of CDs, the researchers reported many precursors and prepared diferent CDs to detect ClO − and perform imaging in vitro and vivo [18][19][20]. Methods for endogenous ClO − detection and measurement in RAW 264.7 cells using nitrogen-fuorinedoped carbon quantum dots (N, F-CDs) are few, and it is necessary to hunt for the precursors and develop the sensitive and efcient sensors. Terefore, designing efcient carbon quantum dots for quantitative analysis of ClO − and selective imaging of ClO − in RAW 264.7 cells is of great signifcance.
Here, N, F-CDs were simply prepared utilizing the novel precursor via one-pot hydrothermal strategy. Te asprepared composites displayed excellent fuorescence properties, good water solubility, low toxicity, and biocompatibility owing to nitrogen and fuorine doping. Additionally, the N, F-CDs exhibited excellent selectivity and high sensitivity for ClO − with efective fuorescence quenching based on their unique performance. Tus, we successfully developed an efective strategy to quantitatively analyze ClO − in real water. In addition, this probe was seamlessly used for the imaging of living cells and sensitive detection of endogenous ClO − in living RAW 264.7 cells.

Instrumentation and Characterization.
A Shimadzu RF-6000 spectrometer (Tokyo, Japan) was used for fuorescence intensity measurements. A UV-5500 spectrophotometer (Shanghai Metash Instruments Co. Ltd., China) recorded the UV-vis absorption spectra at 20°C. Te particle size of N, F-CDs was accurately measured using Tecnai G2F30 instrument (Termo Fisher Scientifc, USA). Te Fourier-transform infrared (FT-IR) spectra were measured using an iS10 FT-IR spectrometer (Nicolet Corporation, USA). Atomic force microscopy (AFM) image was obtained on Dimension Icon (Bruker, Germany). To investigate the N-and F-doping status in N, F-CDs, X-ray photoelectron spectroscopy (XPS) was carried out using a 250 Xi instrument (Termo Fisher Scientifc). Confocal microscopic images were obtained using UltraVIEW VoX& IX81 (Olympus, Japan) scanning.

Preparation of N, F-CDs.
Ammonium citrate tribasic (243 mg), L-alanine (445 mg), and ammonium fuoride (222 mg) as precursor were added to 10 mL of ultrapure water. Te mixture was carefully mixed by ultrasonication (10 min) and then transferred to a polytetrafuoroethylene-lined autoclave (50 mL) and reacted at 160°C for 4 h in an oven. Subsequently, the resulting solution was naturally cooled to room temperature (25°C ± 10°C) and purifed using a 0.22 μm end remover flter to remove large particles. , and MnO 4 − were added to 1.96 mL of N, F-CD solution (0.094 mg/mL). Ten, the fuorescence spectrum was measured under an excitation wavelength of 356 nm after 5 min.
Te sensitivity of ClO − was investigated in 1.96 mL of N, F-CD solution (0.094 mg/mL). In a typical assay, the fuorescence data of N, F-CDs with diferent concentrations of ClO − (0.00, 1.00, 6.25, 12.5, 25.0, 50.0, 100, and 600 μM) were investigated. Te emission spectrum of the above solution was recorded under excitation wavelength of 356 nm. All measurements were repeated fve times.

Real Sample Assays.
Te feasibility and practicality of the prepared N, F-CDs-based probe were tested by generalizing the detection of diferent water samples. Lake, tap, and swimming pool water samples were collected from Wanfeng Lake (Xingyi, China), Minzu Normal University of Xingyi (Xingyi, China), and its gymnasium (Xingyi, China), respectively. All raw samples were fltered through a 0.22 μm end remover flter to remove the large particles. Te concentration of ClO − in the water samples was detected using the developed probe method. In brief, 40 μL of the water sample was added into the N, F-CD solution (0.094 mg/mL, 1.96 mL), fuorescence intensity was measured at excitation wavelength of 356 nm, and the reliability of the developed method was further assessed via the spiked recovery approach.

Cytotoxicity and Cellular
Imaging. Before cellular imaging of RAW 264.7 cells, the MTT assay was used to evaluate the potential cytotoxicity of N, F-CDs for RAW 264.7 cells. Te details are as follows: RAW 264.7 cells were incubated and treated with diferent concentrations (15.6-1000 μg/mL) of N, F-CDs at 37°C for 24 h. Afterwards, the MTT reagent (20 μL, 5 mg/mL) was added to each hole, and the cells were incubated at 37°C for 4 h. Finally, 150 μL dimethyl sulfoxide was added to each hole to dissolve and crystallize the precipitates. Cell survival rate was computed as the ratio of cells in the solution treated with the probe to those in the control group.
To explore the potential application of N, F-CDs, mouse macrophage-like cell line RAW 264.7 was cultured in HyClone with fetal bovine serum (10%, w/w) at 37°C under 5% CO 2 atmosphere. Ten, the cells were transferred to new confocal dishes and divided into fve groups. Tree groups were incubated with multifarious concentrations (50, 100, and 200 μg/mL) of the N, F-CDs (3 h) and normal saline (4 h) in each well. Meanwhile, the remaining two groups of cells were stimulated by PMA (2 μg/mL) and LPS (100 μg/ mL) for 4 h; then, the cell groups were cultivated with N, F-CDs for 3 h to detect the endogenous ClO − . Te group incubated with 200 μM N, F-CDs was added to each hole and cultivated for 3 h. Subsequently, NaOCl (50 μM) was added for another 4 h to detect the exogenous ClO − . Ten, phosphate-bufered saline solution (pH � 7.4) was used to wash the cells for three times. Te fuorescence of stained cells was observed, and the images were taken in blue channels (370 nm).

Optimization of Preparation Conditions for N, F-CDs.
Te preparation conditions including reaction time, reaction temperature, and diluted concentration were studied ( Figure S1). As shown in Figure S1A, the fuorescence intensity increased with the reaction time from 2-4 h. After 4 h, the fuorescence intensity of N, F-CDs gradually decreased. In consequence, 4 h was selected as the optimal reaction time. Figure S1B demonstrates that the N, F-CDs illustrate the best fuorescence properties below 160°C. Finally, Figure S1C shows the strongest fuorescence intensity with a raw N, F-CD solution at 80-fold dilution (1.14 mg/ mL).

Characterization of N, F-CDs.
Te morphology of N, F-CDs was characterized by transmission electron microscopy (TEM) and AFM ( Figure 1). Te TEM images display that the prepared N, F-CDs are spherical composites with an average diameter of 2.9 nm and a narrow particle size distribution of 2.3-3.5 nm (Figure 1(b)). Most particles are amorphous carbon, as can been seen in the high-resolution TEM image. Te interplanar spacing of lattice fringes is 0.203 nm, corresponding to the (100) facets of graphitic carbon [21]. As shown in Figures 1(c) and 1(d), the AFM images are consistent with the TEM results, showing an average height of N, F-CDs of approximately 2.9 nm.
To reveal the surface-functional groups of N, F-CDs, the FT-IR and XPS survey spectra were adopted. As shown in Figure S2A, the wide absorption at 3107 cm −1 is attributed to the stretching vibrations of O-H [22]. Moreover, the small peaks at 1593 and 1455 cm −1 originate from the stretching vibration band of C�C and C-N groups [23], respectively. Te peaks at 1014 and 1255 cm −1 are consistent with the bending vibrations of the C-O and C-F groups [24], respectively. Te characteristic peaks at 1408 and 744 cm −1 are attributed to C�N and -NH [25], respectively. Moreover, the XPS survey spectrum, which is further used to investigate the surface state and composition of N, F-CDs, displays the peaks at 284.8, 401.0, 531.9, and 685.6 eV, which are ascribed to C1s, N1s, O1s, and F1s, respectively ( Figure S2B). Te elemental analysis results of N, F-CDs revealed the atomic ratios of C, O, N, and F to be 60.22%, 28.18%, 10.45%, and 1.15%, respectively. Tis fnding indicates considerable doping percentages of F and N.
Te high-resolution spectrum of C1s (Figure 2(a)) signal exhibits three peaks at 284.8 (C-C/C-N), 285.8 (C-O), and 288.9 eV (C-F). As depicted in Figure 2(b), the two peaks at 399.5 (C-N�C) and 401.6 eV (N-H) appeared in the highresolution N1s spectrum. Te O1s spectrum (Figure 2(c)) demonstrates three ftted peaks at 531.7 eV, 532.4, and, 533.9 eV, which are attributed to the C�O, C-O, and C-OH groups, respectively. Te F1s spectrum (Figure 2(d)) peaks at 685.7 and 686.7 eV correspond to the semi-ionic C-F and covalent C-F bonds [24,26], respectively. Te XPS results are consistent with the FT-IR results. Hydrophilic functional groups such as -NH 2 , C-OH, and O�C-OH on the surface of N, F-CDs impart excellent water solubility [27].

Optical Properties of N, F-CDs.
Te N, F-CDs were distilled with water (1.14 mg/mL), and then their optical properties were explored. It is important to explore the excitation and emission wavelengths of N, F-CDs for their potential applications. Te excitation wavelength was approximately 300-390 nm, and the emission band was concentrated at approximately 350-550 nm (Figure 3(a)). Te maximum excitation peak was observed at 356 nm, while the maximum emission peak was observed at 440 nm. Tese fndings revealed the typical fuorescence dependence of N, F-CDs between the excitation and emission wavelengths. Te aqueous solution of N, F-CDs (Figure 3(b), left inset) presented strong blue fuorescence emission (right) under 356 nm. Te UV-vis spectra of N, F-CDs show the characteristic peaks at 340 nm, which can be attributed to the n-π * transition of the N, F-CDs core due to the presence of C�O, C-F, and N-H groups on their surfaces [28].
Fluorescence stability plays a crucial role in quantitative analysis. As shown in Figure S3A, the N, F-CD fuorescence intensity did not change within 24 h, proving the excellent fuorescence stability of N, F-CDs. Te photoluminescence response of N, F-CDs was studied by adding NaOH and HCl to adjust the pH to diferent levels. As illustrated in Figure S3B, the fuorescence intensity of N, F-CDs was closely related to pH; when the pH was 1.0-4.0, the fuorescence intensity increased gradually; within the wide pH range of 4.0-14.0, the fuorescence intensity did not change signifcantly, which meant that this range was suitable for application. Moreover, quinine sulfate (QY = 54% in 0.1 M H 2 SO 4 at 360 nm) was used as the standard sample to calculate the fuorescence quantum yield (QY) of N, F-CDs. Te average QY of the N, F-CDs in aqueous solution was 26.3% at 24°C ± 4°C.  (Figure 4(a)). Furthermore, the fuorescence intensity gradually decreased with increasing concentration of ClO − from 2.5 μM to 600 μM (Figure 4(b)), and the quenched relationship could be quantifed using the linear correlation in the following equation:

Fluorescence Measurement of ClO
where F 0 and F are the fuorescence intensities of N, F-CDs at 356 nm in the absence and presence of ClO − , respectively, and C is the concentration of ClO − . Te linear relationship was 2.50-600 μM with a correlation coefcient R 2 of 0.996. Te limit of detection was calculated to be (S/N = 3, n = 5) 0.75 μM. Te developed probe method clearly demonstrated that N, F-CDs can be used to detect trace ClO − amounts and evidenced their promising applications in environmental and biomedical systems.

Detecting ClO − in Water Samples.
To explore the practical applications of N, F-CDs, the developed method was used to detect trace amounts of ClO − in water samples obtained from Wanfeng Lake, tap, and a swimming pool. As shown in Table 1, the recovery ranged from 92.2% to 120%, with relative standard deviations (RSDs) of less than 12%.
Tese results indicate that the N, F-CDs-based probing method was accurate and reliable. Moreover, the proposed method demonstrated that the probe can be used to detect ClO − in diferent water samples.
Medicines (a)    the lifetime, fuorescence quenching can be attributed to the dynamic mode. Tese results were further verifed by the UV-vis absorption spectra in Figure 5(b). After adding ClO − to the N, F-CD solution, the absorption intensity markedly decreased at 340 nm. Te decrease process revealed that ClO − may selectively oxidate the amino N groups on the surface of N, F-CDs to form new substances with less π-π and n-π conjugate systems at 300-600 nm, thereby leading to the fuorescence quenching of N, F-CDs. Tese results proved that a dynamic quenching mode occurred between N, F-CDs and ClO − [29,30].

Cytotoxicity Assays and Cell Imaging.
To assess the cytotoxicity of N, F-CDs and develop their potential application in bioimaging, traditional MTT assays were employed to test their cytotoxicity in RAW 264.7. As expected, more than 83% of the RAW 264.7 cells were viable after exposure at 500 μg·mL −1 ( Figure S4). Te results exhibit excellent properties of the N, F-CDs, such as low toxicity and excellent biocompatibility, indicating their potential use as biomarkers.
Te practicality and feasibility of N, F-CDs were further evaluated in cell imaging. As shown in Figure 6, the obtained confocal image of RAW 264.7 cells became brighter with increasing concentration of the probe. Te overlaid image reveals that N, F-CDs can easily penetrate cell membranes or translocate by endocytosis. Moreover, when the bright N, F-CDs (200 μg/mL) were added with LPS (100 μg/mL) and PMA (2 μg/mL) or NaOCl (50 μM), the intracellular fuorescence confocal intensity of RAW 264.7 cells weakened because of the intracellular presence of trace ClO − . Terefore, these experimental results further prove that the N, F-CDs probes can track native ClO − and the fuctuations of endogenous/exogenous ClO − levels in live RAW 264.7 cells.

Conclusions
Novel N, F-CDs (QY � 26.3%) were successfully synthesized using ammonium citrate tribasic, L-alanine, and ammonium fuoride via a facile, low-cost, ecofriendly hydrothermal approach. Te as-prepared N, F-CDs are nanometer-sized (2.9 nm) CDs, exhibiting excellent highly fuorescent stability, low biotoxicity, water solubility, and biocompatibility. Tus, the broad application prospects of N, F-CDs were demonstrated. First, N, F-CDs can function as highly selective and sensitive fuorescent probes for ClO − . Second, they can be used for quantitatively detecting ClO − in real water samples, ofering a low detection limit of 0.75 μM and broad linear range of 2.50-600 μM. Finally, owing to their low biotoxicity, water solubility, and biocompatibility, N, F-CDs can be used in in vitro imaging. Te results show that the probes can not only exhibit cell permeability but can also efectively detect endogenous/exogenous ClO − in living RAW 264.7 cells. Tis probe is expected to provide a new approach for detecting ClO − in other organelles.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest. photoelectron spectroscopy (XPS) spectrum. Figure S3: (A) fuorescence stability of time; (B) efect of pH. Figure S4: N, F-CD MTT assay of RAW 264.7 (n = 3). (Supplementary  Materials)